1. We investigated the effects of acetylcholine (Ach) on identified motoneurons (MNs) using an in vitro preparation of the crayfish thoracic nervous system. Discontinuous current-clamp and single electrode voltage- clamp recordings from 50 MNs were performed along with micropipette pressure ejection of Ach (or agonists) close to the recording electrode. 2. Localized ejections of relatively large volumes (500-2,500 pl) of Ach (10-2 M) or of the muscarinic agonist oxotremorine (Oxo, 10-2 M) onto the MN neuropile region, usually (90% of the cases) induced a slow, alternating rhythmic activity in antagonistic MNs. In other cases (4 experiments), with similar deliveries of Ach or Oxo, MNs developed the ability to fire rhythmically but only when depolarized by sustained current injection. Pressure ejections of smaller volumes (50-200 pl) of Ach (10-2 M) close to the recorded MN could give rise to a fast (1-2 s) large amplitude (≤20 mV) membrane depolarization (12%), a long-lasting (10 s to several minutes) and small (2-5 mV) depolarization (14%), and a combination of the two (74%). These responses appeared to involve different regions of the neurite because they changed when the drug-ejection pipette was displaced in the neuropile. Moreover, fast and long-lasting depolarizing components resulted from a direct effect of Ach onto the MNs because they persisted under tetrodotoxin (TTX, 10-6 M) and cobalt (Co2+, 5 x 10-3 M) superfusion. 3. Whereas the membrane resistance decreased during the fast Ach-induced depolarization, it increased during the long-lasting depolarization. The increase in membrane resistance was more pronounced at depolarized potentials more than -55 mV and involve a reduction in K+ conductance. 4. Superfusion with nicotinic and muscarinic antagonists revealed that the fast Ach-induced depolarization involved nicotinic receptors, muscarinic receptors, or both, whereas the slow depolarization was exclusively muscarinic. 5. The Ach-evoked inward currents were studied under voltage clamp. The fast nicotinic component (I(nic)) increased with hyperpolarizing holding potentials and decreased with depolarizing potentials, reversing at between 10 and 30 mV. The fast muscarinic current (I(fmus)) displayed similar characteristics and reversed at about -10 mV. Whereas both fast components were voltage independent, the long-lasting muscarinic component (I(smus)) was voltage dependent. The response grew with membrane depolarization, but when the holding potential was hyperpolarized below resting level, the response declined to disappear at about -60 mV and beyond. 6. The long-lasting cholinergic response involved the inhibition of a voltage-dependent K+ conductance, which was tonically active at potentials positive to -60 mV and that resulted in outward rectification around resting and depolarized levels. Evidence is provided indicating that the long- lasting muscarinic conductance plays a key role in the generation of rhythmic activity in the walking system.